Method of producing erythrocytes

ABSTRACT

Provided herein are methods of producing erythrocytes from hematopoietic cells, particularly hematopoietic cells from placental perfusate in combination with hematopoietic cells from umbilical cord blood, wherein the method results in accelerated expansion and differentiation of the hematopoietic cells to more efficiently produce administrable erythrocytes. Further provided herein is a bioreactor in which hematopoietic cell expansion and differentiation takes place.

This application claims benefit of U.S. Provisional Application No.60/963,894, filed Aug. 6, 2007, the disclosure of which is herebyincorporated by reference in its entirety.

1. FIELD

Provided herein, generally, are methods of expanding hematopoietic cellpopulations, e.g., CD34⁺ cell populations, and methods of producingadministrable units of erythrocytes from such cell populations. Alsoprovided herein is a bioreactor that accomplishes such expansion anddifferentiation.

2. BACKGROUND

Each year in the United States approximately 13 million units of bloodare used for transfusion or to generate life-saving blood products suchas platelets. Voluntary blood donation is utilized by the Red Cross andother agencies to procure from about 500 mL to about 1000 mL whole bloodsamples. Self-screening of voluntary donation is relatively safe andeffective in the US and Western Europe where the incidences of HIV andother adventitious pathogens are relatively low. However, in countriesin which HIV and hepatitis are endemic, procurement of safe blood fortransfusion can be highly problematic. As an alternative to voluntaryblood donation many groups have attempted to develop safe artificialblood substitutes that could undergo long-term storage. While some ofthese products show significant promise in transiently treatingtraumatic blood loss, such products are not designed for long-termsubstitution of red blood cell function. Increasingly there is a need todevelop a safe and plentiful supply of erythrocytes that can beadministered to patients on the battlefield or civilian hospitalsettings around the world.

Conventional methods for producing erythrocytes are either inefficient,too small in scale, or too laborious to allow for the continuous,on-site production of erythrocytes. Conventional dish or flask-basedculture systems are associated with discontinuous medium exchange, andgenerally dish-based culture systems cannot be used to handle singlebatches of >10⁹ cells. A logical further development from dishes are bagtechnologies, e.g. the Wave Bioreactor, in which the medium volume issignificantly enlarged by using bags and cell attachment surface can beenlarged by using buoyant carriers. However, bag-type reactors typicallyoperate from 2×10⁶ to about 6×10⁶ cells/ml medium, requiring significantmedia dilution during culture and a laborious 10-100 fold debulking.Moreover, bag technologies, and generally all large-vessel stirred tanktype bioreactors, do not provide tissue-like physiologic environmentsthat are conducive to “normal” cell expansion and differentiation.

3. SUMMARY

Provided herein are methods of expanding hematopoietic cells (e.g.,hematopoietic stem cells or hematopoietic progenitor cells), todifferentiating the expanded hematopoietic cells into administrableerythrocytes (red blood cells), and to the production of administrableunits of cells comprising the erythrocytes.

In one aspect, provided herein is a method of producing erythrocytes. Inone embodiment, the method comprises differentiating hematopoietic cellsfrom human placental perfusate to erythrocytes. In a specificembodiment, the method comprises expanding a population of hematopoieticcells in the absence of a feeder layer, wherein the hematopoietic cellsare obtained from human placental perfusate and optionally from humanumbilical cord blood; subsequently expanding said hematopoietic cells inthe presence of a feeder layer; and differentiating the hematopoieticcells to erythrocytes or progenitors of erythrocytes.

In another aspect, provided herein is a bioreactor for the expansion ofhematopoietic cells and differentiation of said hematopoietic cells intoerythrocytes. The bioreactor allows for production of a number oferythrocytes equivalent to current methods of producing erythrocytes, ina much smaller volume, by facilitating a continuous erythrocyteproduction method rather than a batch method. In specific embodiments,the bioreactor comprises a cell culture element, a cell separationelement, a gas provision element and/or a medium provision element. In aspecific embodiment of the bioreactor, the erythrocytes are collected bymagnetic bead separation. In another embodiment of the method, theerythrocytes are collected by partially or fully deoxygenatinghemoglobin in said erythrocytes, and attracting the erythrocytes to asurface using a magnetic field.

In another aspect, provided herein is a method of the production oferythrocytes using the bioreactor described herein. In a specificembodiment, provided herein is a method of producing erythrocytescomprising producing erythrocytes using a plurality of the bioreactorsdisclosed herein. In other specific embodiments of the method, theproduction of said erythrocytes is automated.

In one aspect, provided herein is a method of producing erythrocytes,comprising (a) expanding a plurality of hematopoietic cells in theabsence of feeder cells, optionally in contact with an immunomodulatorycompound, wherein the immunomodulatory compound increases the number ofhematopoietic cells compared to a plurality of hematopoietic cells notin contact with the immunomodulatory compound, to produce a firstexpanded hematopoietic cell population; (b) expanding the first expandedhematopoietic cell population in the presence of a plurality of feedercells to produce a second expanded hematopoietic cell population; (c)contacting said second expanded hematopoietic cell population with oneor more factors that cause differentiation of hematopoietic cells insaid second expanded hematopoietic cell population into erythrocytes;and (d) isolating said erythrocytes from said second expandedhematopoietic cell population. In a specific embodiment, said isolatingof erythrocytes in step (d) is performed continuously. In other specificembodiments, said isolating of erythrocytes in step (d) is performedperiodically, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, ormore. In another specific embodiment, said isolating of erythrocytes instep (d) is performed periodically when one or more culture conditioncriteria are met, e.g., achievement in the culture of a particular celldensity; achievement in the culture of a particular number of cells permilliliter expressing certain erythrocyte markers, e.g., CD36 orglycophorin A; or the like.

In a specific embodiment, said hematopoietic cells are CD34⁺. In anotherspecific embodiment, said hematopoietic cells are Thy-1⁺, CXCR4⁺, CD133⁺or KDR⁺. In another specific embodiment, said hematopoietic cells areCD45⁻. In another specific embodiment, said hematopoietic cells areHLA-DR⁻, CD71⁻, CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD19⁻, CD16⁻, CD24⁻,CD56⁻, CD66b⁻ and/or glycophorin A⁻. In another specific embodiment,said hematopoietic stem cells are Lin⁻.

In another specific embodiment, said hematopoietic cells are obtainedfrom cord blood, placental blood, peripheral blood, or bone marrow. Inanother specific embodiment, said hematopoietic cells are obtained fromplacental perfusate. In another specific embodiment, said hematopoieticcells are obtained from umbilical cord blood and placental perfusate. Ina more specific embodiment, said placental perfusate is obtained bypassage of perfusion solution through only the vasculature of aplacenta. In another specific embodiment, said hematopoietic cells arehuman hematopoietic cells.

In another specific embodiment, said feeder cells are from the sameindividual as the hematopoietic cells. In another specific embodiment,said feeder cells are from a different individual as the hematopoieticcells. In a more specific embodiment, said feeder cells are adherentplacental stem cells, bone marrow-derived mesenchymal stem cells,mesenchymal stem cells from peripheral blood, mesenchymal stem cellsfrom cord blood, or stromal stem cells, or a combination of any of theforegoing. In another specific embodiment, said feeder cells areadherent placental stem cells. In a more specific embodiment, saidadherent placental stem cells are CD200⁺ or HLA-G⁺; CD73⁺, CD105⁺, andCD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said stem cells whensaid population is cultured under conditions that allow formation ofembryoid-like bodies; or OCT-4⁺ and facilitate formation of one or moreembryoid-like bodies in a population of isolated placental cellscomprising said stem cell when cultured under conditions that allowformation of embryoid-like bodies. In another more specific embodiment,the adherent placental stem cells are CD10⁺, CD34⁻, CD105⁺, and CD200⁺.In another more specific embodiment, the adherent placental stem cellsare HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻. In another more specificembodiment, the adherent placental stem cells are CD10⁺, CD13⁺, CD33⁺,CD45⁻, CD117⁻ and CD133⁻. In another more specific embodiment, theadherent placental stem cells are CD10⁻, CD33⁻, CD44⁺, CD45⁻, andCD117⁻. In another more specific embodiment, the adherent placental stemcells are HLA A,B,C⁺, CD45⁻, CD34⁻, CD133⁻; positive for CD10, CD13,CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117.In another more specific embodiment, the adherent placental stem cellsare CD200⁺ and CD10⁺, as determined by antibody binding, and CD117⁻, asdetermined by both antibody binding and RT-PCR. In another more specificembodiment, the adherent placental stem cells are CD10⁺, CD29⁻, CD54⁺,CD200⁺, HLA-G⁺, HLA class I⁺ and β-2-microglobulin⁺.

In certain embodiments of the method, a plurality of said hematopoieticcells is blood type A, blood type O, blood type AB, blood type O; is Rhpositive or Rh negative; blood type M, blood type N, blood type S, orblood type s; blood type P1; blood type Lua, blood type Lub, or bloodtype Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−)or K− k− Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+); blood typeFy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) orJk(a+b+). In a more specific embodiment, the hematopoietic cells aretype O, Rh positive; type O, Rh negative; type A, Rh positive; type A,Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rhpositive or type AB, Rh negative. In other specific embodiments of themethod, greater than 90%, 95%, 98%, or 99%, or each, of saidhematopoietic cells is blood type A, blood type O, blood type AB, bloodtype O; is Rh positive or Rh negative; blood type M, blood type N, bloodtype S, or blood type s; blood type P1; blood type Lua, blood type Lub,or blood type Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+),Kp(a−b−) or K− k− Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+);blood type Fy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−),Jk(a−b+) or Jk(a+b+). In more specific embodiments, the hematopoieticcells are type O, Rh+; type O, Rh negative; type A, Rh positive; type A,Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rhpositive or type AB, Rh negative.

As used herein, the term “hematopoietic cells” includes hematopoieticstem cells and hematopoietic progenitor cells, that is, blood cells ableto differentiate into erythrocytes.

As used herein, “+”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is detectably present influorescence activated cell sorting over an isotype control; or isdetectable above background in quantitative or semi-quantitative RT-PCR.

As used herein, “−”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is not detectablypresent in fluorescence activated cell sorting over an isotype control;or is not detectable above background in quantitative orsemi-quantitative RT-PCR.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Flow cytometric analysis of HPP-derived CD34⁺/CD45⁻ andCD34⁺/CD45⁺ cells.

FIG. 2: Cell expansion in pomalidomide supplemented IMDM medium. FIG.2A: Fold expansion of total nucleated cells (TNC). FIG. 2B: Foldexpansion of CD34⁺ cells.

FIG. 3: CFU-forming activity of expanded cultures. FIG. 3A: Foldexpansion of TNC. FIG. 3B: Fold expansion of CD34⁺ cells. FIG. 3C:Expansion of CD34⁺ cells in number of cells. “Compound 1” ispomalidomide.

FIG. 4: Ex vivo expansion of CD34⁺ cells from HPP (FIG. 4A) andumbilical cord blood (UCB) (FIG. 4B) in cytokine-supplemented RPMIMedium (see Example 3).

FIG. 5: Giemsa staining of CD34⁺ cultures in cytokine supplemented RPMImedium. FIG. 5A: CD34⁺ cells at day 0, showing medium blue color. FIG.5B: Basophilic normoblasts at day 9, showing medium blue color. FIG. 5C:Orthochromatophilic normoblasts at day 11, showing medium blue color.FIG. 5D: Polychromatophilic erythrocytes/erythrocytes at day 21, showingpink color. All images were obtained at magnification 400×.

5. DETAILED DESCRIPTION

Provided herein is a method of producing erythrocytes from expandedhematopoietic cells, e.g., hematopoietic stem cells and/or hematopoieticprogenitor cells. In one embodiment, hematopoietic cells are collectedfrom a source of such cells, e.g., placental perfusate and umbilicalcord blood. The hematopoietic cells are expanded first without the useof feeder cells. Such isolation and expansion can be performed in acentral facility, which provides expanded hematopoietic cells forshipment to decentralized expansion and differentiation at points ofuse, e.g., hospital, military base, military front line, or the like.Expansion at point-of-use, in a preferred embodiment, is accomplishedusing feeder cells. Feeder cell-dependent expansion, according to themethods in Section 5.2.2, below, preferably takes place within abioreactor, as exemplified herein. Differentiation of erythrocytes,according to the methods of Section 5.3, below, also preferably takesplace in the bioreactor. Collection of erythrocytes produced in themethod, in a preferred embodiment, is performed continuously orperiodically, e.g., during differentiation. The continuous or periodicseparation aspect of the method allows for the production oferythrocytes in a substantially smaller space than possible using, e.g.,batch methods. The time for collection and expansion of thehematopoietic cells is approximately 5-10 days, typically about 7 days.Erythrocytes are purified on-site and packaged into administrable units.

In one aspect, provided herein is a method of producing erythrocytes,comprising (a) expanding a plurality of hematopoietic cells in theabsence of feeder cells, optionally in contact with an immunomodulatorycompound, wherein the immunomodulatory compound increases the number ofhematopoietic cells compared to a plurality of hematopoietic cells notin contact with the immunomodulatory compound, to produce a firstexpanded hematopoietic cell population; (b) expanding the first expandedhematopoietic cell population in the presence of a plurality of feedercells to produce a second expanded hematopoietic cell population; (c)contacting said second expanded hematopoietic cell population with oneor more factors that cause differentiation of hematopoietic cells insaid second expanded hematopoietic cell population into erythrocytes;and (d) isolating said erythrocytes from said second expandedhematopoietic cell population. In a specific embodiment, said isolatingof erythrocytes in step (d) is performed continuously. In other specificembodiments, said isolating of erythrocytes in step (d) is performedperiodically, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, ormore. In another specific embodiment, said isolating of erythrocytes instep (d) is performed periodically when one or more culture conditioncriteria are met, e.g., achievement in the culture of a particular celldensity; achievement in the culture of a particular number of cells permilliliter expressing certain erythrocyte markers, e.g., CD36 orglycophorin A; or the like.

5.1. Hematopoietic Cells

Hematopoietic cells useful in the methods disclosed herein can be anyhematopoietic cells able to differentiate into erythrocytes, e.g.,precursor cells, hematopoietic progenitor cells, hematopoietic stemcells, or the like. Hematopoietic cells can be obtained from tissuesources such as, e.g., bone marrow, cord blood, placental blood,peripheral blood, or the like. Hematopoietic cells can be obtained fromplacenta. In a specific embodiment, the hematopoietic cells are obtainedfrom placental perfusate. Hematopoietic cells from placental perfusatecan comprise a mixture of fetal and maternal hematopoietic cells, e.g.,a mixture in which maternal cells comprise greater than 5% of the totalnumber of hematopoietic cells. Preferably, hematopoietic cells fromplacental perfusate comprise at least about 90%, 95%, 98%, 99% or 99.5%fetal cells.

In certain embodiments, the hematopoietic cells are CD34⁺ cells. CD34⁺hematopoietic cells can, in certain embodiments, express or lack thecellular marker CD38. Thus, in specific embodiments, the hematopoieticcells useful in the methods disclosed herein are CD34⁺CD38⁺ orCD34⁺CD38⁻. In a more specific embodiment, the hematopoietic cell isCD34⁺CD38⁻Lin⁻. In another specific embodiment, the hematopoietic cellis one or more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻,CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In another specific embodiment,the hematopoietic cell is CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻,CD19⁻, CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In another more specificembodiment, the hematopoietic cell is CD34⁺CD38⁻CD33⁻CD117⁻. In anothermore specific embodiment, the hematopoietic cell isCD34⁺CD38⁻CD33⁻CD117⁻ CD235⁻CD36⁻.

In another embodiment, the hematopoietic cells are CD45⁻. In a specificembodiment, the hematopoietic cells are CD34⁺CD45⁻. In another specificembodiment, the hematopoietic cells are CD34⁺CD45⁺.

In another embodiment, the hematopoietic cell is Thy-1⁺. In a specificembodiment, the hematopoietic cell is CD34⁺Thy-1⁺. In anotherembodiment, the hematopoietic cells are CD133⁺. In specific embodiments,the hematopoietic cells are CD34⁺CD133⁺ or CD133⁺Thy-1⁺. In anotherspecific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁺. Inanother specific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁻.In another embodiment, the hematopoietic cells are positive for KDR(vascular growth factor receptor 2). In specific embodiments, thehematopoietic cells are CD34⁺KDR⁺, CD133⁺KDR⁺ or Thy-1⁺KDR⁺. In certainother embodiments, the hematopoietic cells are positive for aldehydedehydrogenase (ALDH⁺), e.g., the cells are CD34⁺ALDH⁺.

In certain embodiments, the hematopoietic cells are CD34⁻.

The hematopoietic cells can also lack certain markers that indicatelineage commitment, or a lack of developmental naiveté. For example, inanother embodiment, the hematopoietic cells are HLA-DR⁻. In specificembodiments, the hematopoietic cells are CD34⁺HLA-DR⁻, CD133⁺HLA-DR⁻,Thy-1⁺HLA-DR⁻ or ALDH⁺HLA-DR⁻ In another embodiment, the hematopoieticcells are negative for one or more, preferably all, of lineage markersCD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b andglycophorin A.

Thus, populations of hematopoietic cells can be selected for use in themethods disclosed herein on the basis of the presence of markers thatindicate an undifferentiated state, or on the basis of the absence oflineage markers indicating that at least some lineage differentiationhas taken place. Methods of isolating cells on the basis of the presenceor absence of specific markers is discussed in detail, e.g., in Section5.1.2, below.

Hematopoietic cells used in the methods provided herein can be asubstantially homogeneous population, e.g., a population comprising atleast about 95%, at least about 98% or at least about 99% hematopoieticcells from a single tissue source, or a population comprisinghematopoietic cells exhibiting the same hematopoietic cell-associatedcellular markers. For example, in various embodiment, the hematopoieticcells can comprise at least about 95%, 98% or 99% hematopoietic cellsfrom bone marrow, cord blood, placental blood, peripheral blood, orplacenta, e.g., placenta perfusate.

Hematopoietic cells used in the methods provided herein can be obtainedfrom a single individual, e.g., from a single placenta, or from aplurality of individuals, e.g., can be pooled. Where the hematopoieticcells are obtained from a plurality of individuals and pooled, it ispreferred that the hematopoietic cells be obtained from the same tissuesource. Thus, in one embodiment, the pooled hematopoietic cells are allfrom placenta, e.g., placental perfusate, all from placental blood, allfrom umbilical cord blood, all from peripheral blood, and the like.

Hematopoietic cells used in the methods disclosed herein can comprisehematopoietic cells from two or more tissue sources. Preferably, whenhematopoietic cells from two or more sources are combined for use in themethods herein, a plurality of the hematopoietic cells used to produceerythrocytes comprise hematopoietic cells from placenta, e.g., placentaperfusate. In various embodiments, the hematopoietic cells used toproduce erythrocytes comprise hematopoietic cells from placenta and fromcord blood; from placenta and peripheral blood; form placenta andplacental blood, or placenta and bone marrow. In a preferred embodiment,the hematopoietic cells comprise hematopoietic cells from placentalperfusate in combination with hematopoietic cells from cord blood,wherein the cord blood and placenta are from the same individual, i.e.,wherein the perfusate and cord blood are matched. In embodiments inwhich the hematopoietic cells comprise hematopoietic cells from twotissue sources, the hematopoietic cells from the sources can be combinedin a ratio of, for example, 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3,9:2, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1 or 9:1.

Preferably, the erythrocytes produced from hematopoietic cells accordingto the methods provided herein are homogeneous with respect to bloodtype, e.g., identical with respect to cell surface markers, antigens, orthe like. Such homogeneity can be achieved, for example, by obtaininghematopoietic cells from a single individual of the desired blood type.In embodiments in which hematopoietic cells are pooled from a pluralityof individuals, it is preferred that each of the individuals shares atleast one, at least two, or at least three or more antigenic blooddeterminants in common. In various embodiments, for example, theindividual from which the hematopoietic cells are obtained is, or eachof the individuals from which hematopoietic cells are obtained are,blood type O, blood type A, blood type B, or blood type AB. In otherembodiments, the individual from which the hematopoietic cells areobtained is, or each of the individuals from which hematopoietic cellsare obtained are, Rh positive, or Rh negative. In a specific embodiment,the individual from which the hematopoietic cells are obtained is, oreach of the individuals from which hematopoietic cells are obtained are,O positive and Rh negative. In more specific embodiments, the individualfrom which the hematopoietic cells are obtained is, or each of theindividuals from which hematopoietic cells are obtained are, O positive,O negative, A positive, A negative, B positive, B negative, AB positive,or AB negative. In other specific embodiments, the individual from whichthe hematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type M, blood type N,blood type S, or blood type s. In other specific embodiments, theindividual from which the hematopoietic cells are obtained is, or eachof the individuals from which hematopoietic cells are obtained are,blood type P1. In other specific embodiments, the individual from whichthe hematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Lua, blood typeLub, or blood type Lu(a). In other specific embodiments, the individualfrom which the hematopoietic cells are obtained is, or each of theindividuals from which hematopoietic cells are obtained are, blood typeK (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K− k− Kp(a−b−). Inother specific embodiments, the individual from which the hematopoieticcells are obtained is, or each of the individuals from whichhematopoietic cells are obtained are, blood type Le(a−b−), Le(a+b−) orLe(a−b+). In other specific embodiments, the individual from which thehematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Fy a, Fy b orFy(a−b−). In other specific embodiments, the individual from which thehematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Jk(a−b−),Jk(a+b−), Jk(a−b+) or Jk(a+b+). In other specific embodiments, theindividual from whom the hematopoietic cells are obtained isclassifiable within blood group Diego, Cartwright, Xgm Scianna,Bombrock, Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich,Cromer, Knops, Indian, Ok, Raph, or JMH. In other specific embodiments,each of the individuals from which hematopoietic cells are obtained areof the same blood type within a blood typing system or group ofantigenic determinants, wherein said blood typing system or group ofantigenic determinants are Diego, Cartwright, Xgm Scianna, Bombrock,Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich, Cromer, Knops,Indian, Ok, Raph, or JMH.

5.1.1. Placental Hematopoietic Stem Cells

In certain embodiment, the hematopoietic cells used in the methodsprovided herein are placental hematopoietic cells. As used herein,“placental hematopoietic cells” means hematopoietic cells obtained fromthe placenta itself, and not from placental blood or from umbilical cordblood. In one embodiment, placental hematopoietic cells are CD34⁺. In aspecific embodiment, the placental hematopoietic cells are predominantly(e.g., at least about 90%, 95% or 98%) CD34⁺CD38⁻ cells. In anotherspecific embodiment, the placental hematopoietic cells are predominantly(e.g., at least about 90%, 95% or 98%) CD34⁺CD38⁺ cells. Placentalhematopoietic cells can be obtained from a post-partum mammalian (e.g.,human) placenta by any means known to those of skill in the art, e.g.,by perfusion.

In another embodiment, the placental hematopoietic cell is CD45⁻. In aspecific embodiment, the hematopoietic cell is CD34⁺CD45⁻. In anotherspecific embodiment, the placental hematopoietic cells are CD34′CD45⁺.

5.1.1.1. Obtaining Placental Hematopoietic Cells by Perfusion

Placental hematopoietic cells can be obtained using perfusion. Methodsof perfusing mammalian placenta to obtain cells, including placentalhematopoietic cells, are disclosed, e.g., in U.S. Pat. No. 7,045,148,entitled “Method of Collecting placental Stem Cells,” U.S. Pat. No.7,255,879, entitled “Post-Partum Mammalian Placenta, Its Use andPlacental Stem Cells Therefrom,” and in U.S. Application No.2007/0190042, entitled “Improved Medium for Collecting Placental StemCells and Preserving Organs,” the disclosures of which are herebyincorporated by reference in their entireties.

Placental hematopoietic cells can be collected by perfusion, e.g.,through the placental vasculature, using, e.g., a saline solution (forexample, phosphate-buffered saline, a 0.9% NaCl solution, or the like),culture medium or organ preservation solution as a perfusion solution.In one embodiment, a mammalian placenta is perfused by passage ofperfusion solution through either or both of the umbilical artery andumbilical vein. The flow of perfusion solution through the placenta maybe accomplished using, e.g., gravity flow into the placenta. Preferably,the perfusion solution is forced through the placenta using a pump,e.g., a peristaltic pump. The umbilical vein can be, e.g., cannulatedwith a cannula, e.g., a TEFLON® or plastic cannula, that is connected toa sterile connection apparatus, such as sterile tubing, which, in turnis connected to a perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid through the placentalvasculature and surrounding tissue. The placenta can also be perfused bypassage of a perfusion fluid into the umbilical vein and collection fromthe umbilical arteries, or passage of a perfusion fluid into theumbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilicalvein are connected simultaneously, e.g., to a pipette that is connectedvia a flexible connector to a reservoir of the perfusion solution. Theperfusion solution is passed into the umbilical vein and artery. Theperfusion solution exudes from and/or passes through the walls of theblood vessels into the surrounding tissues of the placenta, and iscollected in a suitable open vessel, e.g., a sterile pan, from thesurface of the placenta that was attached to the uterus of the motherduring gestation. The perfusion solution may also be introduced throughthe umbilical cord opening and allowed to flow or percolate out ofopenings in the wall of the placenta which interfaced with the maternaluterine wall. Placental cells that are collected by this method, whichcan be referred to as a “pan” method, are typically a mixture of fetaland maternal cells.

In another embodiment, the perfusion solution is passed through theumbilical veins and collected from the umbilical artery, or is passedthrough the umbilical artery and collected from the umbilical veins.Placental cells collected by this method, which can be referred to as a“closed circuit” method, are typically almost exclusively fetal.

The closed circuit perfusion method can, in one embodiment, be performedas follows. A post-partum placenta is obtained within about 48 hoursafter birth. The umbilical cord is clamped and cut above the clamp. Theumbilical cord can be discarded, or can processed to recover, e.g.,umbilical cord stem cells, and/or to process the umbilical cord membranefor the production of a biomaterial. The amniotic membrane can beretained during perfusion, or can be separated from the chorion, e.g.,using blunt dissection with the fingers. If the amniotic membrane isseparated from the chorion prior to perfusion, it can be, e.g.,discarded, or processed, e.g., to obtain stem cells by enzymaticdigestion, or to produce, e.g., an amniotic membrane biomaterial, e.g.,the biomaterial described in U.S. Application Publication No.2004/0048796.

After cleaning the placenta of all visible blood clots and residualblood, e.g., using sterile gauze, the umbilical cord vessels areexposed, e.g., by partially cutting the umbilical cord membrane toexpose a cross-section of the cord. The vessels are identified, andopened, e.g., by advancing a closed alligator clamp through the cut endof each vessel. The apparatus, e.g., plastic tubing connected to aperfusion device or peristaltic pump, is then inserted into each of theplacental arteries. The pump can be any pump suitable for the purpose,e.g., a peristaltic pump. Plastic tubing, connected to a sterilecollection reservoir, e.g., a blood bag such as a 250 mL collection bag,is then inserted into the placental vein. Alternatively, the tubingconnected to the pump is inserted into the placental vein, and tubes toa collection reservoir(s) are inserted into one or both of the placentalarteries. The placenta is then perfused with a volume of perfusionsolution, e.g., about 750 ml of perfusion solution. Cells in theperfusate are then collected, e.g., by centrifugation.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental hematopoieticcells may vary depending upon the number of hematopoietic cells to becollected, the size of the placenta, the number of collections to bemade from a single placenta, etc. In various embodiments, the volume ofperfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mLto 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or750 mL to 2000 mL. Typically, the placenta is perfused with 700-800 mLof perfusion liquid following exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days to obtain placental hematopoietic cells.Where the placenta is to be perfused a plurality of times, it may bemaintained or cultured under aseptic conditions in a container or othersuitable vessel, and perfused with a stem cell collection composition(see U.S. Application Publication No. 2007/0190042, the disclosure ofwhich is incorporated herein by reference in its entirety), or astandard perfusion solution (e.g., a normal saline solution such asphosphate buffered saline (“PBS”)) with or without an anticoagulant(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/orwith or without an antimicrobial agent (e.g., β-mercaptoethanol (0.1mM); antibiotics such as streptomycin (e.g., at 40-100 μg/ml),penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 μg/ml). Inone embodiment, an isolated placenta is maintained or cultured for aperiod of time without collecting the perfusate, such that the placentais maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or moredays before perfusion and collection of perfusate. The perfused placentacan be maintained for one or more additional time(s), e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or more hours, and perfused a second time with, e.g., 700-800 mLperfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or moretimes, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferredembodiment, perfusion of the placenta and collection of perfusionsolution, e.g., stem cell collection composition, is repeated until thenumber of recovered nucleated cells falls below 100 cells/ml. Theperfusates at different time points can be further processedindividually to recover time-dependent populations of cells, e.g.,placental hematopoietic cells. Perfusates from different time points canalso be pooled.

5.1.1.2. Obtaining Placental Hematopoietic Cells by Tissue Disruption

Hematopoietic cells can be isolated from placenta by perfusion with asolution comprising one or more proteases or other tissue-disruptiveenzymes (e.g., trypsin, collagenase, papain, chymotrypsin, subtilisin,hyaluronidase; a cathepsin, a caspase, a calpain, chymosin, plasmepsin,pepsin, or the like). In a specific embodiment, a placenta or portionthereof (e.g., amniotic membrane, amnion and chorion, placental lobuleor cotyledon, umbilical cord, or combination of any of the foregoing) isbrought to 25-37° C., and is incubated with one or moretissue-disruptive enzymes in 200 mL of a culture medium for 30 minutes.Cells from the perfusate are collected, brought to 4° C., and washedwith a cold inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and2 mM beta-mercaptoethanol. The stem cells are washed after severalminutes with cold (e.g., 4° C.) stem cell collection composition.

In one embodiment, the placenta can be disrupted mechanically (e.g., bycrushing, blending, dicing, mincing or the like) to obtain thehematopoietic cells. The placenta can be used whole, or can be dissectedinto components prior to physical disruption and/or enzymatic digestionand hematopoietic cell recovery. For example, hematopoietic cells can beobtained from the amniotic membrane, chorion, umbilical cord, placentalcotyledons, or any combination thereof.

Placental hematopoietic cells can also be obtained by enzymaticdisruption of the placenta using a tissue-disrupting enzyme, e.g.,trypsin, collagenase, papain, chymotrypsin, subtilisin, hyaluronidase; acathepsin, a caspase, a calpain, chymosin, plasmepsin, pepsin, or thelike. Enzymatic digestion preferably uses a combination of enzymes,e.g., a combination of a matrix metalloprotease and a neutral protease,for example, a combination of collagenase and dispase. In oneembodiment, enzymatic digestion of placental tissue uses a combinationof a matrix metalloprotease, a neutral protease, and a mucolytic enzymefor digestion of hyaluronic acid, such as a combination of collagenase,dispase, and hyaluronidase or a combination of LIBERASE (BoehringerMannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymesthat can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental stem cells. For example, inone embodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the placental hematopoietic cells.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental hematopoietic cells collected will comprise a mix of placentalstem cells derived from both fetal and maternal sources. Where a portionof the placenta that comprises no, or a negligible number of, maternalcells (for example, amnion), the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.1.2. Isolation, Sorting, and Characterization of Cells

Cells, including hematopoietic cells from any source, e.g., mammalianplacenta, can initially be purified from (i.e., be isolated from) othercells by Ficoll gradient centrifugation. Such centrifugation can followany standard protocol for centrifugation speed, etc. In one embodiment,for example, cells collected from the placenta are recovered fromperfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in, e.g., fresh saline solution, or amedium suitable for stem cell maintenance, e.g., IMDM serum-free mediumcontaining 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY). The totalmononuclear cell fraction can be isolated, e.g., using LYMPHOPREP®(Nycomed Pharma, Oslo, Norway) according to the manufacturer'srecommended procedure.

As used herein, “isolating” cells, including placental cells, e.g.,placental hematopoietic cells or placental stem cells, means to removeat least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of thecells with which the isolated cells are normally associated in theintact tissue, e.g., mammalian placenta. A cell from an organ is“isolated” when the cell is present in a population of cells thatcomprises fewer than 50% of the cells with which the stem cell isnormally associated in the intact organ.

Adherent placental cells obtained by perfusion or digestion, e.g., foruse as feeder cells, can, for example, be further, or initially,isolated by differential trypsinization using, e.g., a solution of 0.05%trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differentialtrypsinization is possible because placental stem cells typically detachfrom plastic surfaces within about five minutes whereas other adherentpopulations typically require more than 20-30 minutes incubation. Thedetached placental stem cells can be harvested following trypsinizationand trypsin neutralization, using, e.g., Trypsin Neutralizing Solution(TNS, Cambrex). In one embodiment of isolation of adherent cells,aliquots of, for example, about 5-10×10⁶ cells are placed in each ofseveral T-75 flasks, preferably fibronectin-coated T75 flasks. In suchan embodiment, the cells can be cultured with commercially availableMesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed in atissue culture incubator (37° C., 5% CO₂). After 10 to 15 days,non-adherent cells are removed from the flasks by washing with PBS. ThePBS is then replaced by MSCGM. Flasks are preferably examined daily forthe presence of various adherent cell types and in particular, foridentification and expansion of clusters of fibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34, in an assay such as an ELISA or RIA, or byFACS; if so, the cell is CD34⁺. Likewise, if a cell, e.g., a feedercell, e.g., an adherent placental stem cell, produces enough OCT-4 RNAto be detectable by RT-PCR, or significantly more OCT-4 RNA than anadult cell, the cell is OCT-4⁺. Antibodies to cell surface markers(e.g., CD markers such as CD34) and the sequence of stem cell-specificgenes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using fluorescence activated cell sorting (FACS). FACS is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In one embodiment, stem cells from placenta are sorted, e.g., isolated,on the basis of expression one or more of the markers CD34, CD38, CD44,CD45, CD73, CD105, CD117, CD200, OCT-4 and/or HLA-G.

In another embodiment, hematopoietic cells, e.g., CD34⁺, CD133⁺, KDR⁺ orThy-1⁺ cells, are sorted, e.g., isolated, on the basis of markerscharacteristic of undifferentiated hematopoietic cells. Such sorting canbe done, e.g., in a population of cells that has not been sorted, e.g.,a population of cells from a perfusion or a tissue digestion, whereinCD34⁺ cells represent a minority of the cells present in the population.Such sorting can also be done in a population of cells that is mostly(e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%)hematopoietic cells as, for example, a purification step. For example,in a specific embodiment, CD34⁺ cells, KDR⁺ cells, Thy-1⁺ cells, and/orCD133⁺ cells are retained during sorting to produce a population ofundifferentiated hematopoietic cells.

In another embodiment, cells, e.g., hematopoietic cells are sorted,e.g., excluded, on the basis of markers of lineage-differentiated cells.For example, cells, in a population of hematopoietic cells, that areCD2⁺, CD3⁺, CD11b⁺, CD11c⁺, CD14⁺, CD16⁺, CD19⁺, CD24⁺, CD56⁺, CD66b⁺and/or glycophorin A⁺ are excluded during sorting from the population ofhematopoietic cells to produce a population of undifferentiatedhematopoietic cells.

In another embodiment, hematopoietic cells can be sorted, e.g.,isolated, on the basis of lack of expression of, e.g., lineage markers.In a specific embodiment, for example, hematopoietic cells, e.g., CD34⁺cells, can be isolated based on a determination that the cells are oneor more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻, CD24⁻,CD56⁻, CD66b⁻ and/or glycophorin A⁻.

In another embodiment, cells, e.g., adherent placental stem cells, aresorted first on the basis of their expression of CD34, wherein CD34⁻cells are retained. In a specific embodiment, CD34⁻ cells that areCD200⁺HLA-G⁺ are separated from all other CD34⁻ cells. In anotherembodiment, cells from placenta are based on their expression of markersCD200 and/or HLA-G; for example, cells displaying either of thesemarkers are isolated for further use. Cells that express, e.g., CD200and/or HLA-G can, in a specific embodiment, be further sorted based ontheir expression of CD73 and/or CD105, or epitopes recognized byantibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45.For example, in one embodiment, placental cells are sorted byexpression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38and CD45, and placental cells that are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺,CD34⁻, CD38⁻ and CD45⁻ are isolated from other placental cells forfurther use.

In another embodiment, magnetic beads can be used to separate cells,e.g., DYNABEADS® (Invitrogen). The cells may be sorted using a magneticactivated cell sorting (MACS) technique, a method for separatingparticles based on their ability to bind magnetic beads (0.5-100 μmdiameter). A variety of useful modifications can be performed on themagnetic microspheres, including covalent addition of antibody thatspecifically recognizes a particular cell surface molecule or hapten.The beads are then mixed with the cells to allow binding. Cells are thenpassed through a magnetic field to separate out cells having thespecific cell surface marker. In one embodiment, these cells can thenisolated and re-mixed with magnetic beads coupled to an antibody againstadditional cell surface markers. The cells are again passed through amagnetic field, isolating cells that bound both the antibodies. Suchcells can then be diluted into separate dishes, such as microtiterdishes for clonal isolation.

Adherent placental stem cells, e.g., to be used as feeder cells, canalso be characterized and/or sorted based on cell morphology and growthcharacteristics. For example, placental stem cells can be characterizedas having, and/or selected on the basis of, e.g., a fibroblastoidappearance in culture. Placental stem cells can also be characterized ashaving, and/or be selected, on the basis of their ability to formembryoid-like bodies. In one embodiment, for example, placental cellsthat are fibroblastoid in shape, express CD73 and CD105, and produce oneor more embryoid-like bodies in culture are isolated from otherplacental cells. In another embodiment, OCT-4⁺ placental cells thatproduce one or more embryoid-like bodies in culture are isolated fromother placental cells.

In another embodiment, placental stem cells, e.g., placentalhematopoietic cells or adherent placental stem cells, can be identifiedand characterized by a colony forming unit assay. Colony forming unitassays are commonly known in the art, such as MESEN CULT™ medium (StemCell Technologies, Inc., Vancouver British Columbia)

Placental stem cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.2. Expansion of Hematopoietic Cells

Once a population of hematopoietic cells is obtained, the population isexpanded. One unit of erythrocytes is expected to comprise about1-2×10¹² red blood cells. Hematopoietic stem cell population doublingrequires approximately 36 hours. Thus, starting from about 5×10⁷hematopoietic cells according to standard methods, and assuming 100%efficiency in expansion and differentiation, production of a unit oferythrocytes would require approximately 14 hematopoietic cellpopulation doublings, or approximately 3 weeks. The method described indetail below improves on standard methods by improving the cultureconditions of hematopoietic cells and increasing the number ofhematopoietic cells during expansion per unit time.

5.2.1. Shortened Hematopoietic Cell Expansion Time

Cells, including hematopoietic cells, comprise cell cycle controlmechanisms, which include cyclins and cyclin-dependent kinases (CDKs),that control the rate of cell division. Cell cycle checkpoints are usedby cells to monitor and regulate the progress of the cell cycle. If acell fails to meet the requirements of a phase it will not be allowed toproceed to the next phase until the requirements have been met. Theprocesses associated with qualifying the cell for progression throughthe different phases of the cell cycle (checkpoint regulation) arerelatively slow and contribute to the relatively modest rate of celldivision observed in mammalian cells, even under optimal in vitroculture conditions.

In one embodiment of the method of producing erythrocytes, the methoduses hematopoietic cells that have a reduced population doubling time.In a specific embodiment, the hematopoietic cells are modified toexpress higher-than-normal levels of a cell cycle activator, or alower-than-normal level of a cell cycle inhibitor, wherein theengineered cells have a detectably shorter doubling time than unmodifiedhematopoietic cells. In a more specific embodiment, the hematopoieticcells are modified to express a higher-than-normal level of one or moreof the cell cycle activator cyclin T2 (CCNT2), cyclin T2B (CCNT2B),CDC7L1, CCN1, cyclin G (CCNG2), cyclin H (CCNH), CDKN2C, CDKN2D, CDK4,cyclin D1, cyclin A, cyclin B, Hes1, Hox genes and/or FoxO.

In another more specific embodiment, the hematopoietic cells express alower-than-normal level of CDK inhibitors p21, p27 and/or TReP-132.Reduction of expression of CDK inhibitors can be accomplished by anymeans known in the art, e.g., the use of small molecule inhibitors,antisense oligonucleotides targeted to a p21, p27 and/or TReP-132 DNA,pre-mRNA or mRNA sequence, RNAi, or the like.

Modifications of hematopoietic progenitor cells in the context of thepresent method of producing erythrocytes are expected to be safe in atherapeutic context, as erythrocytes are enucleated and incapable ofreplication.

In another specific embodiment, the hematopoietic cells are modified toexpress higher-than-normal levels of a cell cycle activator, wherein theengineered cells have a detectably shorter doubling time than, ordetectably increased rate of proliferation compared to, unmodifiedhematopoietic cells, and where the increased expression of a cell cycleactivator is inducible. Any inducible promoter known in the art can beused to construct such a modified hematopoietic cell, e.g., atetracycline-inducible gene expression system using a stably expressedreverse tetracycline-controlled transactivator (rtTA) under the controlof a CMV promoter (e.g., REVTET-ON® System, Clontech Laboratories, PaloAlto, Calif.); U.S. Patent Application Publication No. 2007/0166366“Autologous Upregulation Mechanism Allowing Optimized Cell Type-Specificand Regulated Gene Expression Cells”; and U.S. Patent ApplicationPublication No. 2007/0122880 “Vector for the Inducible Expression ofGene Sequences,” the disclosure of each of which is incorporated hereinby reference in its entirety.

Expression of a gene encoding a cell cycle inhibitor or negative cellcycle regulator can be disrupted in a hematopoietic cell, e.g., byhomologous or non-homologous recombination using standard methods.Disruption of expression of a cell cycle inhibitor or negative cellcycle regulator can also be effected, e.g., using an antisense moleculeto, e.g., p21, p27 and/or TReP-132.

In another embodiment, hematopoietic cells used to produce erythrocytesare modified to express notch 1 ligand such that expression of the notch1 ligand results in detectably decreased senescence of the hematopoieticcells compared to unmodified hematopoietic cells; see Berstein et al.,U.S. Patent Application Publication 2004/0067583 “Methods forImmortalizing Cells,” the disclosure of which is incorporated herein byreference in its entirety.

In another specific embodiment, the medium in which the hematopoieticcells are expanded enhance faithful DNA replication, e.g., the mediumincludes one or more antioxidants.

In a preferred embodiment, the method of producing erythrocytes includesa step that excludes any modified hematopoietic cells, orpre-erythrocyte precursors, from the final population of isolatederythrocytes produced in the method disclosed herein. Such separationcan be accomplished as described elsewhere herein on the basis of one ormore markers characteristic of hematopoietic cells not fullydifferentiated into erythrocytes. The exclusion step can be performedsubsequent to an isolation step in which erythrocytes are selected onthe basis of erythrocyte-specific markers, e.g., CD36 and/or glycophorinA.

5.2.2. Feeder Cell-Independent Expansion of Hematopoietic Cells

In certain embodiments, hematopoietic cells used in the methods providedherein are expanded in culture without the use of a feeder layer toproduce a population of expanded hematopoietic cells to produce, e.g., afirst expanded hematopoietic cell population. The hematopoietic cellscan be expanded by any feeder cell-independent method known to those ofskill in the art. In one embodiment, feeder-free expansion ofhematopoietic cells is the first of at least two expansion steps priorto differentiation of the hematopoietic cells into erythrocytes.

Feeder cell-independent expansion of hematopoietic cells can take placein any container compatible with cell culture and expansion, e.g.,flask, tube, beaker, dish, multiwell plate, bag or the like. In aspecific embodiment, feeder cell-independent expansion of hematopoieticcells takes place in a bag, e.g., a flexible, gas-permeable fluorocarbonculture bag (for example, from American Fluoroseal). In a specificembodiment, the container in which the hematopoietic cells are expandedis suitable for shipping, e.g., to a site such as a hospital or militaryzone wherein the expanded hematopoietic cells are further expanded anddifferentiated, e.g., using the bioreactor described below.

Hematopoietic cells, in certain embodiments, are expanded in a culturemedium comprising one or more cytokines or growth factors. In oneembodiment, the medium in which the hematopoietic cells are expandedcomprise one or more of Flt-3 ligand, thrombopoietin, and stem cellfactor (SCF). In a specific embodiment, hematopoietic cells at about2×10⁴ cells per milliliter are expanded in contact with about 50 ng/mLFlt-3 ligand, about 100 ng/mL thrombopoietin, and about 100 ng/mL SCF.Expansion times can range, e.g., from about 3 days to about 21 days,e.g., about 7 days.

In one embodiment, hematopoietic cells are expanded by culturing saidcells in contact with an immunomodulatory compound, e.g., a TNF-αinhibitory compound, for a time and in an amount sufficient to cause adetectable increase in the proliferation of the hematopoietic cells overa given time, compared to an equivalent number of hematopoietic cellsnot contacted with the immunomodulatory compound. See, e.g., U.S. PatentApplication Publication No. 2003/0235909, the disclosure of which ishereby incorporated by reference in its entirety. In a preferredembodiment, the immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisolindoline-1′-one)-1-piperidine-2,6-dione;4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;4-amino-2-[(3RS)-2,6-dioxopiperidin-3-yl]-2H-isoindole-1,3-dione;α-(3-aminophthalimido) glutarimide; pomalidomide, lenalidomide, orthalidomide. In another embodiment, said immunomodulatory compound is acompound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another embodiment, saidimmunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another embodiment, said immunomodulatory compound is a compoundhaving the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In a specific, preferred embodiment, expansion of hematopoietic cells isperformed in IMDM supplemented with 20% BITS (BSA, recombinant humaninsulin and transferrin), SCF, Flt-3 ligand, IL-3, and4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (10 μM in0.05% DMSO). In a more specific embodiment, about 5×10⁷ hematopoieticcells, e.g., CD34⁺ cells, are expanded in the medium to from about5×10¹⁰ cells to about 5×10¹² cells, which are resuspended in 100 mL ofIMDM to produce a population of expanded hematopoietic cells. Thepopulation of expanded hematopoietic cells is preferably cryopreservedto facilitate shipping.

The expanded hematopoietic cells produced without feeder cells asdescribed above, e.g., the first expanded hematopoietic cell population,can be further expanded using feeder cells to produce a second expandedhematopoietic cell population, as described in the following Section.

5.2.3. Expansion Using Feeder Layers

In another embodiment, the hematopoietic cells used in the methodsprovided herein, e.g., a first expanded hematopoietic cell populationproduced by non-feeder cell-dependent expansion of hematopoietic cellsas described above, can be cultured and expanded using a layer of feedercells, e.g., to produce a second expanded hematopoietic cell population.In one embodiment, expansion of the hematopoietic cells in the presenceof a feeder cell layer is the second of at least two hematopoietic cellexpansion steps prior to differentiation of the hematopoietic cells toerythrocytes. Though not wishing to be bound by theory, expansion withfeeder cells is expected to be “asymmetric,” that is, expansion producesa combination of expansion of relatively undifferentiated hematopoieticcells and relatively more differentiated, lineage-committed cells.Lineage-committed cells can, in certain embodiments, be removed duringthe expansion phase.

The feeder cells can be any feeder cells used or known to be useful inthe art. Feeder cells can be of the same species as the hematopoieticcells, or of a different species. In certain embodiments, feeder cellsare from the same individual as the hematopoietic cells, e.g., thefeeder cells and hematopoietic cells are matched. Feeder cells, like thehematopoietic cells, can be autologous to a particular recipient. Feedercell types useful for culturing hematopoietic cells can be, e.g., bonemarrow-derived mesenchymal stem cells (e.g., see U.S. Pat. No.5,486,359), tissue culture plastic-adherent placental stem cells,mesenchymal-like stem cells from cord blood, placental blood orperipheral blood, adult stem cells, or the like). In another embodiment,the feeder cells are fully-differentiated cells, e.g., fibroblasts, suchas human skin fibroblasts or mouse embryonic fibroblasts, or humanumbilical vein endothelial cells.

In certain embodiments, the feeder cells are not treated to reduceproliferation or differentiation. In certain other embodiments, thefeeder layer cells are treated with, e.g., mitomycin C or ionizingradiation, e.g., gamma radiation, to suppress or prevent proliferationof the feeder cells, e.g., about 100 cGy to about 100 Gray, e.g., 1 Grayto about 60 Gray. In a specific embodiment, the cells are treated with1500 cGy ionizing radiation to produce feeder cells. Methods ofirradiating feeder cells can be found in, e.g., U.S. ApplicationPublication No. 2006/0223183. In a specific embodiment, the feeder cellsare cryopreserved prior to irradiation. In another specific embodiment,a plurality of human fibroblasts are irradiated with from about 1 Grayto about 60 Gray, e.g., about 1.5 Gray, ionizing radiation to produce aplurality of feeder cells.

In one embodiment, the feeder cells and hematopoietic cells are culturedin a bioreactor, e.g., a bioreactor substantially as described elsewhereherein. In a specific embodiment of the bioreactor, the hematopoieticcells and feeder cells are co-cultured, that is, such that a pluralityof the hematopoietic cells are in physical contact with a plurality ofthe feeder cells. In another embodiment, the hematopoietic cells arecultured separately, wherein culture medium from the feeder cells iscirculated to the hematopoietic cells. In a specific embodiment, thehematopoietic cells expanded using a feeder layer are hematopoieticcells that were expanded with an immunomodulatory compound prior toculture with or contact with said feeder layer.

5.2.4. Adherent Placental Stem Cells as Feeder Cells

As noted above, adherent placental stem cells can be used as feedercells in the methods provided herein. Adherent placental stem cells aredescribed in, and are obtainable by the methods disclosed in, U.S. Pat.Nos. 7,045,148 and 7,255,879, and in United States Patent ApplicationPublication Nos. 2007/0275362, and 2008/0032401, the disclosures of eachof which are incorporated herein in their entireties.

Adherent placental stem cells useful in the methods provided hereinexpress a plurality of markers that can be used to identify and/orisolate the stem cells, or populations of cells that comprise the stemcells. The adherent placental stem cells, and stem cell populationsthereof (that is, two or more placental stem cells) include stem cellsand stem cell-containing cell populations obtained directly from theplacenta, or any part thereof (e.g., amnion, chorion, placentalcotyledons, and the like). Adherent placental stem cell populations alsoincludes populations of (that is, two or more) placental stem cells inculture, and a population in a container, e.g., a bag. The adherentplacental stem cells described herein as usable as feeder layers forhematopoietic cell expansion are not trophoblasts, cytotrophoblasts,embryonic stem cells or embryonic germ cells.

The adherent placental stem cells described herein generally express themarkers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do not expressCD34, CD38, or CD45. Placental stem cells can also express HLA-ABC(MHC-1) and HLA-DR. These markers can be used to identify adherentplacental stem cells, and to distinguish adherent placental stem cellsfrom other stem cell types. Because the placental stem cells can expressCD73 and CD105, they can have mesenchymal stem cell-likecharacteristics. However, because the placental stem cells can expressCD200 and HLA-G, a fetal-specific marker, they can be distinguished frommesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells, which express neither CD200 nor HLA-G. In the same manner, thelack of expression of CD34, CD38 and/or CD45 identifies the placentalstem cells as non-hematopoietic stem cells.

Thus, in one embodiment, the feeder cells are isolated adherentplacental stem cells that are CD200⁺ or HLA-G⁺. In another embodiment,the feeder cells are a population of cells comprising, e.g., that isenriched for, adherent CD200⁺, HLA-G⁺ placental stem cells. In anotherembodiment, the feeder cells are isolated adherent placental stem cellsthat are CD73⁺, CD105⁺, and CD200⁺. In another embodiment, the feedercells are an isolated population of cells comprising, e.g., that isenriched for, adherent CD73⁺, CD105⁺, CD200⁺ placental stem cells. Inanother embodiment, the feeder cells are isolated adherent placentalstem cells that are CD200⁺ and OCT-4⁺. In another embodiment, the feedercells are an isolated population of cells comprising, e.g., that isenriched for, adherent CD200⁺, OCT-4⁺ placental stem cells. In anotherembodiment, the feeder cells are isolated adherent placental stem cellsthat are CD73⁺, CD105⁺ and HLA-G⁺. In another embodiment, the feedercells are an isolated population of cells comprising, e.g., that isenriched for, adherent CD73⁺, CD105⁺ and HLA-G⁺ placental stem cells. Inanother embodiment, the feeder cells are isolated adherent placentalstem cells that are CD73⁺ and CD105⁺ and which facilitate the formationof one or more embryoid-like bodies in a population of isolatedplacental cells comprising said stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies.In another embodiment, the feeder cells are a population of isolatedcells comprising, e.g., that is enriched for, adherent CD73⁺, CD105⁺placental stem cells, wherein said population forms one or moreembryoid-like bodies under conditions that allow formation ofembryoid-like bodies. In another embodiment, the feeder cells areisolated adherent placental stem cells that are OCT-4⁺ and whichfacilitate formation of one or more embryoid-like bodies in a populationof isolated placental cells comprising said stem cell when culturedunder conditions that allow formation of embryoid-like bodies. Inanother embodiment, the feeder cells are a population of isolated cellscomprising, e.g., that is enriched for, adherent OCT-4⁺ placental stemcells, wherein said population forms one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, the feeder cells comprise isolated adherentplacental stem cells that are CD10⁺, CD34⁻, CD105⁺, and CD200⁺. In aspecific embodiment of the above embodiments, said stem cells areadditionally CD90⁺ and CD45⁻. In another embodiment, the feeder cellsare isolated adherent placental stem cells that are HLA-A,B,C⁻, CD45⁻,CD133⁻ and CD34⁻. In another embodiment, the feeder cells are isolatedadherent placental stem cell that is CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻and CD133⁻. In another embodiment, the feeder cells are isolatedadherent placental stem cells that are CD10⁻, CD33⁻, CD44⁺, CD45⁻, andCD117⁻. In another embodiment, the feeder cells are isolated adherentplacental stem cells that are HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻, positivefor CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/ornegative for CD117. In another embodiment, feeder cells are isolatedadherent placental stem cells that are CD200⁺ and CD10⁺, as determinedby antibody binding, and CD117⁻, as determined by both antibody bindingand RT-PCR. In another embodiment, the feeder cells are isolatedadherent placental stem cells that are CD10⁺, CD29⁻, CD54⁺, CD200⁺,HLA-G⁺, HLA class I⁺ and β-2-microglobulin⁺.

Adherent placental stem cells used as feeder layers can be freshlyisolated from the placenta, or can have been, for example, passaged atleast once, at least three times, at least five times, at least 10times, at least 15 times, or at least 20 times.

Adherent placental stem cells can be used as feeder cells, for example,by contacting adherent placental stem cells with mitomycin C, orirradiating the cells, for a time sufficient to detectably suppressproliferation of the cells, and plating the cells, e.g., on a tissueculture surface, at about 5,000 cells per cm². The adherent placentalstem cells are allowed to attach to the surface, e.g., for 10 minutes to60 minutes. The plated, attached adherent placental stem cells are thenready to be inoculated with hematopoietic cells for hematopoietic cellculture and expansion.

5.3. Production of Erythrocytes from Hematopoietic Cells

Production of erythrocytes from hematopoietic cells, e.g., the expandedhematopoietic cells described above, is preferably performed in abioreactor, e.g., the bioreactor exemplified elsewhere herein.

Differentiation of hematopoietic cells into erythrocytes and/orpolychromatophilic erythrocytes can be accomplished by contacting thehematopoietic cells with one or more erythrogenic cytokines and/orgrowth factors. In one embodiment, hematopoietic cells are contactedwith one or more of stem cell factor (SCF), erythropoietin (Epo),insulin-like growth factor (IGF-1), FMS-like tyrosine kinase 3 ligand(Flt3-L), and thrombopoietin (Tpo) in an amount and for a timesufficient to cause the differentiation of a plurality of thehematopoietic cells to erythrocytes and/or polychromatophilicerythrocytes. In various specific embodiments, at least 50%, 55%, 60%,65%, 70%. 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hematopoieticcells are differentiated to erythrocytes and/or polychromatophilicerythrocytes.

In a specific embodiment, the hematopoietic cells are contacted withcytokines, e.g., SCF, IGF-1 and erythropoietin in an amount and for atime sufficient to cause differentiation of a plurality of thehematopoietic cells into erythrocytes and/or polychromatophilicerythrocytes. In certain embodiments, the hematopoietic cells arecontacted with one or more cytokines or growth factors in an amount andfor a time sufficient to cause differentiation of a plurality of thehematopoietic cells into erythrocytes and polychromatophilicerythrocytes (also known as reticulocytes, blood reticulocytes,diffusely basophilic erythrocytes, or marrow reticulocytes). In certainother embodiments, the hematopoietic cells are contacted with one ormore cytokines or growth factors in an amount and for a time sufficientto cause differentiation of a plurality of the hematopoietic cells intoa precursor of an erythrocyte, e.g., into colony-formingunits-granulocyte, erythrocyte, monocyte; blast-formingunits-erythrocyte; colony-forming units-erythrocyte, pronormoblasts,basophilic normoblasts, polychromatic normoblasts, orthochromicnormoblasts, or polychromatic erythrocytes (reticulocytes).

The above cytokines can be used in any amount that causesdifferentiation a plurality of the hematopoietic cells intoerythrocytes. Typical amounts of cytokines used, for hematopoietic cellsexpanded for about 7 days from a starting population of, e.g., about1×10⁴ to 2×10⁴ hematopoietic cells per milliliter, are about 50 ng/mLSCF; about 3 units/mL Epo; 50 ng/mL IGF-1; 50 ng/mL Flt3-L; and/or 100ng/mL Tpo in, e.g., RPMI medium. Differentiation of the hematopoieticcells in, e.g. SCF, IGF-1 and Epo can proceed for, e.g., 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, typicallyabout 14 days. Optionally, the differentiated cells can be collected,resuspended to about 5×10⁵ per milliliter in medium, e.g., RPMI mediumcomprising Epo (e.g., at about 3 U/mL), IGF-1 (e.g., at about 20 ng/ml),IL-11 (e.g., at about 20 ng/ml) and IL-3 (e.g., at about 50 ng/ml), andcultured for an additional 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 days,typically about 6 days.

In one embodiment, differentiation of hematopoietic cells, e.g., theexpanded hematopoietic cells described above, can be accomplished byculturing said cells in contact with an immunomodulatory compound, e.g.,a TNF-α inhibitory compound as described above, for a time and in anamount sufficient to cause a detectable increase in the proliferation ofthe hematopoietic cells over a given time, compared to an equivalentnumber of hematopoietic cells not contacted with the immunomodulatorycompound. See, e.g., U.S. Patent Application Publication No.2003/0235909, the disclosure of which is incorporated herein byreference in its entirety.

Differentiation of the hematopoietic cells into erythrocytes can beassessed by detecting erythrocyte-specific markers, e.g., by flowcytometry. Erythrocyte-specific markers include, but are not limited to,CD36 and glycophorin A. Differentiation can also be assessed by visualinspection of the cells under a microscope. The presence of typicalbiconcave cells confirms the presence of erythrocytes. The presence oferythrocytes (including reticulocytes) can be confirmed using a stainfor deoxyribonucleic acid (DNA), such as Hoechst 33342, TO-PRO®-3, orthe like. Nucleated precursors to erythrocytes typically stain positivewith a DNA-detecting stain, while erythrocytes and reticulocytes aretypically negative. Differentiation of hematopoietic cells toerythrocytes can also be assessed by progressive loss of transferringreceptor (CD71) expression and/or laser dye styryl staining duringdifferentiation. Erythrocytes can also be tested for deformabilityusing, e.g., an ektacytometer or diffractometer. See, e.g., Bessis M andMohandas N, “A Diffractometric Method for the Measurement of CellularDeformability,” Blood Cells 1:307 (1975); Mohandas N. et al., “Analysisof Factors Regulating Erythrocyte Deformability,” J. Clin. Invest.66:563 (1980); Groner W et al., “New Optical Technique for MeasuringErythrocyte Deformability with the Ektacytometer,” Clin. Chem. 26:1435(1980). Fully-differentiated erythrocytes have a mean corpuscular volume(MCV) of about 80 to about 108 fL (femtoliters); mean corpuscularhemoglobin (MCH) of about 17 to about 31 pg, and a mean corpuscularhemoglobin concentration (MCHC) of about 23% to about 36%.

5.4. Separation of Erythrocytes from Precursors

Erythrocytes produced by the methods described above are preferablyseparated from hematopoietic cells, and, in certain embodiments, fromprecursors of erythrocytes. Such separation can be effected, e.g., usingantibodies to CD36 and/or glycophorin A. Separation can be achieved byknown methods, e.g., antibody-mediated magnetic bead separation,fluorescence-activated cell sorting, passage of the cells across asurface or column comprising antibodies to CD36 and/or glycophorin A, orthe like. In another embodiment, erythrocyte separation is achieved bydeoxygenating the culture medium comprising the erythrocytes, followedby magnetic separation of deoxygenated erythrocytes from other cells.

Erythrocytes can be continuously separated from a population of cells,e.g., from a second expanded hematopoietic cell population as describedabove, or can be separated at intervals. In certain embodiments, forexample, isolation of erythrocytes is performed, e.g., every 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, orevery 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours, or more, or when one or more culturecondition criteria are met, e.g., achievement in the culture of aparticular cell density; achievement in the culture of a particularnumber of cells per milliliter expressing certain erythrocyte markers,e.g., CD36 or glycophorin A; or the like. Separation of erythrocytesfrom a cell population is preferably performed using a bioreactor, asdescribed below.

5.5. Bioreactor Production of Erythrocytes

In another aspect of the method of producing erythrocytes, hematopoieticcells are expanded using a bioreactor. In one embodiment, hematopoieticcells are expanded in a bioreactor in the presence of feeder cells. Inanother embodiment, hematopoietic cells are differentiated in abioreactor. In a more preferred embodiment, the hematopoietic cells areexpanded in a bioreactor, e.g., in the presence of feeder cells, thendifferentiated in the bioreactor. The bioreactor in which thehematopoietic cells are differentiated can be the same bioreactor inwhich the hematopoietic cells are expanded, or can be a separatebioreactor. In another embodiment, the bioreactor is constructed tofacilitate expansion of the hematopoietic cells entirely in thebioreactor. In another embodiment, the bioreactor is constructed toallow expansion of hematopoietic cells in conjunction with feeder cells.In another embodiment, the bioreactor is constructed so as to physicallyseparate the hematopoietic cells from the feeder cells. In anotherembodiment, the bioreactor is constructed to allow the hematopoieticcells and feeder cells to contact one another.

In another embodiment, the bioreactor is constructed to allow continuousflow of cells in media, enabling the continuous separation ofdifferentiated erythrocytes from remaining cells in the bioreactor. Thecontinuous flow and cell separation allows for the bioreactor to beconstructed in a substantially smaller volume than would bioreactorsusing batch methods of producing erythrocytes. In another embodiment,the bioreactor is constructed to allow periodic, e.g., non-continuousflow of cells in media, enabling the periodic separation ofdifferentiated erythrocytes from remaining cells in the bioreactor. Theperiodic flow and cell separation preferably allows for the bioreactorto be constructed in a substantially smaller volume than wouldbioreactors using batch methods of producing erythrocytes. In specificembodiments, isolation of erythrocytes is performed, e.g., every 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specificembodiment, isolation of erythrocytes is performed periodically when oneor more culture condition criteria are met, e.g., achievement in theculture of a particular cell density; achievement in the culture of aparticular number of cells per milliliter expressing certain erythrocytemarkers, e.g., CD36 or glycophorin A; or the like.

In certain embodiments, the bioreactor is disposable.

In one embodiment, the bioreactor comprises a culturing element and acell separation element. In another embodiment, the bioreactor comprisesa medium gas provision element. In another embodiment, the bioreactorcomprises a cell factor element comprising one or more bioactivecompounds. In another embodiment, the elements of the bioreactor aremodular; e.g., separable from each other and/or independently usable. Inone embodiment, the capacity of the bioreactor is about 100 mL, 200 mL,300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, or about 900 mL, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,45 or 50 liters. In another embodiment, the bioreactor, including allcomponents, occupies about 47 cubic feet or less. In another embodiment,the bioreactor is capable of culturing up to about 10¹⁰, 10¹¹, or about10¹² cells, e.g., hematopoietic cells.

In one embodiment, the culturing element comprises a compartment able toreceive culture medium, e.g., culture medium comprising hematopoieticcells or culture medium comprising feeder cells. The culturing elementcomprises a port that allows for the introduction of media and/orhematopoietic cells for culture. Such a port can be any art-acceptableport for such devices, e.g., a Luer-lock seal port. The culturingelement also comprises one or more ports for the passage of media to thecell separation element. The culturing element optionally furthercomprises a port for the introduction of bioactive compounds into theinterior of the culturing element, e.g., a port that facilitatesconnection of the cell factor element to the culturing element. In aspecific embodiment, hematopoietic cells, including differentiatinghematopoietic cells, in the culturing element are continuouslycirculated in medium to a cell separation element (see below) to isolateerythrocytes and/or polychromatophilic erythrocytes and/or othererythrocyte precursors.

The culturing element, in a specific embodiment, comprises a pluralityof interior surfaces or structures suitable for the culture of feedercells, e.g., stromal cells or adherent placental stem cells. Suchsurfaces can be, e.g., tubes, cylinders, hollow fibers, a poroussubstrate, or the like. The surfaces can be constructed of any materialsuitable for the culture of cells, e.g., tissue culture plastic,flexible pharmaceutical grade plastic, hydroxyapatite, polylactic acid(PLA), polyglycolic acid copolymer (PLGA), polyurethane,polyhydroxyethyl methacrylate, or the like. Hollow fibers typicallyrange from about 100 μm to about 1000 μm in diameter, and typicallycomprise pores that allow passage of molecules no more than about 5 kDa,10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa,90 kDa, 100 kDa, 125 kDa, 150 kDa, 175 kDa, 200 kDa, 150 kDa, 300 kDa,350 kDa, 400 kDa, 450 or 500 kDa.

In one embodiment, the surfaces on which the feeder cells are culturedphysically separate the feeder cells from the hematopoietic cells. Forexample, the surfaces can be hollow fibers in the lumen of which thefeeder cells are grown, while hematopoietic cells are cultured in mediasurrounding the fibers. The surfaces can also comprise a set of stackedmembranes that separate two medium compartments, one for the feederlayers and another for the hematopoietic cells. In another embodiment,the surface on which the feeder cells are grown allows for the directcontact between feeder cells and hematopoietic cells.

The cell separation element comprises at least one port for receivingmedium, comprising cells, from the culturing element. The cellseparation element comprises one or more components that facilitate orenable the separation of at least one type of cell, e.g., erythrocytes,from cells in medium from the culture element. Such separation can beeffected, e.g., using antibodies to CD36 and/or glycophorin A.Separation can be achieved by known methods, e.g., antibody-mediatedmagnetic bead separation, fluorescence-activated cell sorting, passageof the cells across a surface or column comprising antibodies to CD36and/or glycophorin A, or the like. In a specific embodiment, the cellseparation element is connected to the cell culturing element, andmedium comprising hematopoietic cells, differentiating hematopoieticcells and erythrocytes is continually passed through the cell separationelement so as to continually remove cells, e.g., erythrocytes from themedium.

In another embodiment, erythrocyte separation is achieved bydeoxygenating culture medium comprising the erythrocytes, followed bymagnetic attraction of deoxygenated erythrocytes, e.g., to a surface orother point of collection.

In another embodiment, the bioreactor comprises a cell separationelement. The cell separation element can comprise one or more componentsthat enable the separation of one or more non-erythrocytic cells (e.g.,undifferentiated or non-terminally differentiated hematopoietic cells)from erythrocytes in the medium. In certain embodiments, the cellseparation element is able to calculate an approximate number oferythrocytes generated, or is able to alert a user that a sufficientnumber of erythrocytes has been generated to constitute a unit,according to preset user parameters.

The bioreactor, in another embodiment, further comprises a gas provisionelement that provides appropriate gases to the culture environment,e.g., contacts the culture medium with a mixture of 80% air, 15% O₂ and5% CO₂, 5% CO₂ in air, or the like. In another embodiment, thebioreactor comprises a temperature element that maintains the medium,the bioreactor, or both at a substantially constant temperature, e.g.,about 35° C. to about 39° C., or about 37° C. In another embodiment, thebioreactor comprises a pH monitoring element that maintains the mediumat a constant pH, e.g., about pH 7.2 to about pH 7.6, or about pH 7.4.In specific embodiments, the temperature element and/or pH monitoringelement comprises a warning that activates when temperature and/or pHexceed or fall below set parameters. In other specific embodiments, thetemperature element and/or pH monitoring element are capable ofcorrecting out-of-range temperature and/or pH.

In a specific embodiment, the bioreactor comprises a cell separationelement and a gas provision element that provides gases to the cultureenvironment, whereby the gas provision element enables the partial orcomplete deoxygenation of erythrocytes, enabling erythrocyte separationbased on the magnetic properties of the hemoglobin contained therein. Ina more specific aspect, the bioreactor comprises an element that allowsfor the regular, or iterative, deoxygenation of erythrocytes produced inthe bioreactor, to facilitate magnetic collection of the erythrocytes.

In another embodiment, the function of the bioreactor is automated,e.g., controlled by a computer. The computer can be, for example, adesktop personal computer, a laptop computer, a Handspring, PALM® orsimilar handheld device; a minicomputer, mainframe computer, or thelike.

5.6. Erythrocyte Units Produced from Hematopoietic Cells

Erythrocyte units produced according to the methods detailed above cancomprise erythrocytes in any useful number or combination of geneticbackgrounds.

In various embodiments, erythrocyte units produced by the methodsprovided herein comprise at least about, at most about, or about 1×10⁸,5×10⁸, 1××10⁹, 5×10⁹, 1×10¹⁹, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 1×10¹²erythrocytes. In various other embodiments, the erythrocyte unitscomprise at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98% or 99% completely-differentiated erythrocytes. In various otherembodiments, the erythrocyte units comprise less than 60%, 50%, 40%,30%, 20%, 10%, 5%, 2% or 1% erythrocyte precursors of any kind. Incertain embodiments, the erythrocyte units produced by the methodsdescribed herein comprise less than about 60%, about 50%, about 40%,about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about15%, about 14%, about 14%, about 12%, about 11%, about 10%, about 9%,about 8%, about 7%, about 6% or about 5% reticulocytes, or othernon-erythrocytic hematopoietic cells. In another embodiment, the unitcomprises erythrocytes from hematopoietic cells from a singleindividual. In another embodiment, the unit comprises erythrocytesdifferentiated from hematopoietic cells from a plurality of individuals.In another embodiment, the unit comprises erythrocytes fromhematopoietic cells from matched human placental perfusate and cordblood. In another embodiment, substantially all (e.g., greater than 99%)of the erythrocytes in a unit of erythrocytes are type O. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type A. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type B. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type AB. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are Rh positive. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are Rh negative.

Naturally-occurring erythrocytes possess certain characteristics thatallow the flow of blood through capillaries. For example, erythrocytesin the aggregate produce non-Newtonian flow behavior, e.g., theviscosity of blood is highly dependent upon shear rates. Normalerythrocytes are deformable and able to build up aggregates/rouleaux.The deformability of erythrocytes appears to be related to theirlifespan in the blood, about 100-120 days; removal of erythrocytes fromthe blood appears to be related to loss of deformability. Normalaggregability of erythrocytes facilitates the cells' flow through thecapillaries, while abnormally increased or decreased aggregabilitydecreases flow. Thus, in preferred embodiments, units of erythrocytesproduced by the methods disclosed herein are assayed as a part ofquality control, e.g., for one or more characteristics ofnaturally-occurring erythrocytes. In certain embodiments, samples oferythrocytes produced by the methods disclosed herein are suspended innatural or artificial plasma and tested for one or more of viscosity,viscoelasticity, relaxation time, deformability, aggregability,blood/erythrocyte suspension yield stress, and mechanical fragility,using normal blood or normal erythrocytes as a control or comparator. Incertain other embodiments, samples of erythrocytes produced as describedherein are assayed for oxygen carrying capacity and oxygen releasecapacity, using normal blood or an equivalent number ofnaturally-occurring erythrocytes as a control.

6. EXAMPLES 6.1. Example 1 Phenotypic Characterization of CD34⁺ Cellsfrom Human Placental Perfusate (HPP) and Umbilical Cord Blood (UCB)

Umbilical cord blood (UCB) was removed from postpartum placentas underinformed consent. The exsanguinated placentas were then perfused togenerate HPP, as described in U.S. Pat. No. 7,045,148, the disclosure ofwhich is incorporated herein by reference in its entirety. After removalof red blood cells (RBCS) the total nucleated cells (TNCs) werecollected and frozen. This method typically resulted in the collectionof about 1-2.5×10⁹ TNCs, compared to around 500 million TNC isolatedfrom UCB.

Flow cytometric analysis of the TNC isolated from exsanguinatedplacentas indicates a high percentage of CD34⁺ cell population ascompared to conventional umbilical cord blood (UCB) generated cellularproduct. TNC from HPP, collected as above, contains about 2-6% CD34⁺cells, compared to about 0.3-1% of the TNC in UCB.

The flow cytometric analysis of the TNC isolated from HPP indicates thata high percentage of the CD34⁺ cell population is CD45⁻ (FIG. 1).

CD34⁺ cells from HPP were plated in a colony-forming unit assay, and theratio (%) of the burst forming unit-erythroid (BFU-E) to the colonyforming unit-erythroid (CFU-E) was determined, as well as the number ofcolony-forming unit-granulocyte, macrophage (CFU-GM) and the number ofcolony-forming unit-granulocyte, erythrocyte, monocyte (CFU-GEMM) (Table1). The clonogenicity was also assessed (Table 1).

TABLE 1 Cell BFU-E/ CFU- CFU- Clono- Sample Purity CFU-E GM GEMMgenicity Donor 1 88% 50.1% 49.5% 0.4% 23.1% Donor 2 92% 54.1% 44.1% 1.7%26.1% Donor 3 94% 32.7% 60.6% 6.7% 19.7%

The colony-forming unit assay was performed according to themanufacturer's protocol (StemCell Technologies, Inc.). In brief, CD34⁺cell suspensions were placed into a methylcellulose medium supplementedwith stem cell factor (SCF), granulocyte colony-stimulating factor(G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin 3 (IL-3), interleukin 6 (IL-6) and erythropoietin (Epo) at100 cells/plate, 300 cells/plate and 1000 cells/plate. For each celldensity, a triplicate assay was performed followed by incubation for 2to 3 weeks. Colony evaluation and enumeration were performed using lightmicroscopy.

In a separate experiment, the ratio (BFU-E)/(CFU-E) for CD34⁺ cells fromHPP and UCB was 46% and 30%, respectively (based on the average valuefor three donors).

These results suggest that HPP-derived cells contain a higher number ofCD34⁺ cells with increased erythrogenic activity relative to UCB-derivedstem cells.

6.2. Example 2 Expansion of CD34⁺ Hematopoietic Cell Populations

The CD34⁺ cell content of human umbilical cord blood (UCB) units isoften not sufficient to provide for hematopoietic cell transplants inadult patients. Ex-vivo expansion of CD34⁺ cells from UCB is oneapproach to overcome this CD34⁺ cell dose limitation. This Exampledemonstrates expansion of CD34⁺ cells using a specific immunomodulatorydrug, 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione(referred to in this Example as pomalidomide).

The ability of pomalidomide to enhance the expansion of human UCBderived CD34⁺ cells in a short-term serum-free, cytokine supplementedculture system was evaluated. CD34⁺ progenitor cells were enriched fromcryopreserved UCB units to >90% purity and seeded (10⁴ CD34⁺ cells) in 1mL of growth medium, which consists of IMDM plus serum substitute BIT(BSA, recombinant human insulin and transferrin, 20%), in the presenceof SCF (50 ng/mL), Flt-3 ligand (50 ng/mL), and IL-3 (10 ng/mL).Pomalidomide, dissolved in DMSO, was supplemented at 2.7 μg/mL. Theculture was incubated at 37° C., 5% CO₂ for 12 days, with fresh mediumadded at day 7. Pomalidomide-free cultures with or without DMSO (0.05%v/v) were used as controls.

In one experiment, pomalidomide supplementation resulted insignificantly higher CD34⁺ expression in the expanded population withoutimpacting total nucleated cell expansion (200-350 fold). CD34⁺ phenotypein the pomalidomide-expanded population was 40-60%, compared with 10-30%in the control. Additionally, pomalidomide appeared to down-regulateCD38 expression on cultured cells. Pomalidomide-expanded CD34⁺ cellswere primarily CD38 negative (95%) and expressed lower levels of CD133(15% vs. 40% in the control). Pomalidomide-expanded CD34⁺ cellsdemonstrated substantial improvement in cumulative colony forming unitsrelative to expanded controls. In another, similar, experiment,pomalidomide supplementation was confirmed to result in significantlyhigher CD34⁺ expression in the expanded population without impactingtotal nucleated cell expansion (200-350 fold). CD34⁺ phenotype in thepomalidomide-expanded population was 40-60%, compared with 10-30% in thecontrol (FIG. 2). Additionally, pomalidomide appeared to down-regulateCD38 expression on cultured cells. Pomalidomide-expanded CD34⁺ cellswere primarily CD38 negative (97%) and expressed lower levels of CD133(11.5% vs. 32.3% in the control). Pomalidomide-expanded CD34⁺ cellsdemonstrated substantial improvement in cumulative colony forming unitsrelative to expanded controls.

The pomalidomide-based CD34⁺ expansion process was scaled up todemonstrate the production of a larger number of CD34⁺ cells. CD34⁺cells were seeded in 10⁴/mL pomalidomide-supplemented medium in aflexible, gas-permeable fluorocarbon culture bag (American Fluoroseal).After 7 days of incubation, the culture was centrifuged and exchangedwith fresh pomalidomide-supplemented medium at three times the initialvolume. By day 12, TNC and CD34⁺ expansion were 350 (range: 250-700) and200 (range: 100-450) fold, respectively (FIG. 3). Viability was 86%(range: 80-90%) by trypan blue. A total of 20 million CD34⁺ cells wereharvested. These results demonstrate that pomalidomide significantlyenhanced the ex-vivo expansion of placental derived CD34⁺ progenitorsand that the process can produce a sufficient amount of CD34⁺ cells forerythrocytic differentiation.

6.3. Example 3 Feeder Cell-Free Expansion of CD34⁺ Precursors andDifferentiation into Erythrocytes

HPP and UCB cells were generally purified using Ficoll to obtain totalnucleated cells (TNCs). TNCs were then used to isolate CD34⁺ cells usinganti-CD34 beads and RoboSep following the protocol provided by themanufacturer (StemCell Technologies, Inc.) If greater than 80% cellpurity was not achieved, further cell sorting was performed usingFACSAria to achieve a purity greater than 90%. In this experiment, CD34⁺cells were isolated with about 92% purity.

In vitro expansion of CD34⁺ cells (about 1×10⁴ CD34⁺ cells/mL) wasperformed in either Iscove's Modified Dulbecco's Medium (IMDM; GIBCO,Grand Island, N.Y.) or RPMI Medium (Sigma-Aldrich) containing Flt-3L (50ng/mL), SCF (100 ng/mL) and Tpo (100 ng/mL) from day 0 to day 7, andcontinued thereafter in IMDM or RPMI medium containing SCF (50 ng/mL),Epo (3 units/mL) and IGF-I (50 ng/mL) from day 8 to day 14. Total cellnumbers were counted at 8, 9, 12, 15 and 18 days. At day 18 TNCexpansion was 300 fold and cell viability was 90%. At day 14, the cellswere suspended at 2×10⁴ to 5×10⁵ cells/mL in IMDM or RPMI mediumcontaining 3 units/mL Epo, 50 ng/mL IGF-1, 20 ng/mL IL-11 (interleukin11), and 50 ng/mL IL-3 (interleukin 3) (R&D Systems) and cultured to day21. FIG. 4 shows the ex vivo expansion of CD34⁺ cells from HPP (FIG. 4A)and UCB (FIG. 4B) in cytokine supplemented RPMI Medium.

6.4. Example 4 Expansion of Hematopoietic Cell Populations Using FeederLayers

This Example explains the use of adherent placental stem cells inco-culturing system using adherent placental stem cells as a feederlayer.

To induce erythrocytic differentiation of the expanded CD34⁺ cells,5×10⁷ cells were resuspended at 1 to 2×10⁴/ml and cultured in RPMIMedium containing 50 ng/mL Flt3-L, 100 ng/mL Tpo, and 100 ng/mL SCF (R&DSystems) for 7 days. The cells collected on day 7 were resuspended at5×10⁴ to 1×10⁵/mL in RPMI medium containing 50 ng/mL SCF, 3 units/mLEpo, and 50 ng/mL IGF-1 (R&D Systems) and cultured up to 14 days. Tofurther differentiate expanded CD34⁺ cells, the expanded cells collectedon day 14 were resuspended at 2×10⁴ to 5×10⁵/mL in RPMI mediumcontaining 3 units/mL Epo, 50 ng/mL IGF-1 with the presence of adherentplacental stem cells treated with mitomycin C. The adherent placentalstem cells were plated at about 5,000 cells per cm² and allowed toattach to the tissue culture surface. Aliquots of cultures of CD34⁺cells were taken at different time points for morphological analysis byGiemsa staining (FIG. 5). Morphological analysis by Giemsa stainingindicated that a percentage of CD34⁺ expanded cells underwent fullmaturation into polychromatophilic erythrocyte/erythrocytes (FIG. 5D).In a subsequent experiment, 11.8% of HPP CD34⁺ cells fullydifferentiated, and 2.37% of UCB CD34⁺ cells fully differentiated, intopolychromatophilic erythrocyte/erythrocytes as determined by stainingwith TO-PRO®-3, a nuclear stain that detects DNA.

Adherent placental stem cells usable as feeder cells can be obtained bymechanical disruption and enzymatic digestion of placenta as follows.Approximately 1 g tissue samples are incubated in a shaker in 1 mg/mLcollagenase 1A for 1 hour at 37° C. at 100 RPM followed by incubationwith 0.25% trypsin for 30 minutes at 37° C. at 100 RPM. The digestedtissue is washed three times with culture medium prior to transferringwashed cells into 75 cm² tissue culture flasks. The stem cells aremaintained in media consisting of 60% v/v Dulbecco's Modified Eagle'sMedium-Low Glucose (DMEM-LG), 40% v/v MCDB-201, supplemented with 2% v/vfetal calf serum, 1× insulin-transferrin-selenium, 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹ M dexamethasone, 10⁻⁴M ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, plateletderived-growth factor −BB (10 ng/ml), and 100 U penicillin/1000 Ustreptomycin, in a 5% CO₂ and humidified incubator at 37° C.Non-adherent cells are removed 12 to 24 hours post plating and half ofthe medium is exchanged every 3-4 days. Cells are subcultured using0.25% trypsin-EDTA and replated at about 4×10³ cells/cm² for continuedexpansion.

6.5. Example 5 Method and Bioreactor for Generating Units ofErythrocytes

This Example provides a method of producing erythrocytes, and abioreactor that enables the production of units of mature erythrocytes.In this particular example, the bioreactor enables the production ofadministrable units of erythrocytes using a five-step process. In thefirst step, hematopoietic cells, e.g., CD34⁺ cells, are isolated. In thesecond step, the CD34⁺ cells are expanded using an immunomodulatorycompound, e.g., pomalidomide. In the third step, the CD34⁺ cells areexpanded in the bioreactor exemplified herein, in a co-culture withadherent placental stem cells, in conjunction with removal oflineage-committed cells. Fourth, remaining uncommitted hematopoieticcells are differentiated to erythrocytes. Finally, in the fifth step,erythrocytes are isolated and collected into administrable units.

Steps 1 and 2, the isolation and initial expansion of hematopoieticcells, are accomplished as described in Examples 3 and 4, above.

Steps 3 and 4 are accomplished using a bioreactor. The bioreactorcomprises a hollow fiber chamber (1) seeded with placental stem cells(2) and an element for gas provision to the medium (3). The bioreactorfurther comprises a coupled cell sorter/separator element (4) thatallows for the continuous separation of committed hematopoietic cells,fully-differentiated erythrocytes, or both. The cell separation elementcan separate the cells from the hematopoietic cells using, e.g.,magnetic cell separation or fluorescence-activated cell separationtechniques.

To initiate cell culture, approximately 5×10⁷ hematopoietic cells, e.g.,CD34⁺ hematopoietic cells, are inoculated into the bioreactor.

6.6. Example 6 Collection of Erythrocytes

This Example exemplifies several methods of the separation oferythrocytes from other lineage committed cells.

Method 1: Erythrocytes, e.g., erythrocytes collected from the cellseparation element of the bioreactor exemplified herein, and hetastarchsolution are mixed 3:1 (v:v) in a Baxter collection bag and placed in anupright position on a plasma extractor. Erythrocytes sediment after 50to 70 minutes. Non-sedimented cells are forced out by the plasmaextractor. Sedimented erythrocytes left in the bag can be furthercollected by centrifugation at 400 g for 10 minutes. After removing thesupernatant, erythrocytes are resuspended in an appropriate amount ofdesired medium.

Method 2—Immunomagnetic separation: Glycophorin A⁺ cells, e.g.,erythrocytes collected from the cell separation element of thebioreactor exemplified herein, are magnetically labeled with GlycophorinA (CD235a) MicroBeads (Miltenyi Biotech). The cell suspension is thenloaded into a tube which is placed in the magnetic field of an EASYSEP®magnet. The magnetically labeled Glycophorin A⁺ cells are retainedinside the tube, while the unlabeled cells are poured off the tube.After removal of the tube from the magnetic field, the magneticallyretained Glycophorin A⁺ cells can be separated from the magnetic beadsand resuspended in an appropriate amount of desired medium.

Method 3—Flow cytometry cell separation: Erythrocytes, e.g.,erythrocytes collected from the cell separation element of thebioreactor exemplified herein, in 500 μl PBS/FBS with 1 μL Fc Block (1/500). 150 μL of the cell suspension is added to each well of a 96 wellV-bottom dish. 50 μL 1° Ab Master Mix (the mix is a 1/25 dilution ofeach primary Ab in PBS/FBS) is added to the cells. One well is includedwith a combination of isotype controls for setting voltage, as well asone well for each of the primary Ab as single positive controls forsetting compensation. The cells are incubated 60 min at 4° C., thencentrifuged at 1500 RPM for two minutes. The supernatant is discarded.The wells are washed with 200 μL PBS/FBS to each well, and mixed bypipetting up and down. The cells are then immediately spun at 1500 RPM×2min; the supernatant is discarded. 150 μL of secondary Ab (i.e.Streptavidin-TC) Master Mix is added, and incubated 30 min at 4° C.,followed by centrifugation at 1500 RPM for 5 minutes. The pellet isresuspended in 200-500 μL of PBS/FBS and transferred to 5 mL flow tubes.Cells are then separated using a flow cytometer.

Method 4: Medium comprising erythrocytes, in continuous flow between thecell culture element and cell separation element, is deoxygenated byreducing or turning off the supply of oxygen from the gas provisionelement, and turning on a magnet in the cell separation element. Mediumis passed through the cell separation element for a sufficient time forthe magnetic field of the magnet to collect erythrocytes to a surface inthe cell separation element. Once a predetermined number of erythrocytesare collected, or collection has proceeded for a predetermined amount oftime, the medium is reoxygenated, releasing the erythrocytes from thesurface.

6.7. Example 7 Bioreactor for Producing Erythrocytes

This Example describes a bioreactor design that allows for improvedproduction of erythrocytes from hematopoietic cells. The bioreactorcomprises a culturing element that comprises hollow fibers in whichhematopoietic cells and feeder cells are cultured. Hematopoietic cells,e.g., hematopoietic progenitor cells, are supplied in a bag at 5×10⁵cells/dose, where one dose yields one unit of blood. The cells areexpanded in the presence of IMDM medium containing 50 ng/mL SCF, 3units/mL Epo, and 50 ng/mL IGF-1 added through a first port. Gasprovision (5% CO₂ in air) occurs through a second port. The medium inwhich the hematopoietic cells are cultured is supplemented withpomalidomide at 2.7 μg/mL. The cells are cultured in contact with feedercells (adherent placental stem cells) that have been seeded in thehollow fiber element of the bioreactor. During culturing, gas, mediummetabolites and medium pH in the culturing element is monitoredcontinuously, and are replenished or exchanged using a programmablecontrol device as necessary. pH of the medium in the culturing elementis maintained at approximately 7 and the culture temperature ismaintained at about 37° C. Lineage-committed cells (i.e., differentiatedcells) are continuously separated and recovered from the culture mediumusing a cell separation element. The bioreactor is equipped with anindependent power supply to enable operation at a remote site, e.g., asite separate from a site at which hematopoietic cells are initiallyobtained.

The present disclosure, including devices, compositions and methods, isnot to be limited in scope by the specific embodiments described herein.Indeed, various modifications in addition to those described herein willbecome apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior invention.

What is claimed is:
 1. A method of producing erythrocytes, comprising(a) expanding a population of isolated human CD34⁺ hematopoietic stemcells from a plurality of human hematopoietic stem cells in a serum-freemedium in contact with one or more factors in the absence of feedercells, wherein said CD34⁺ hematopoietic stem cells are in contact withpomalidomide or lenalidomide for a time and in an amount sufficient forthe pomalidomide or lenalidomide to increase the number of CD34⁺hematopoietic stem cells compared to a population of human CD34⁺hematopoietic stem cells not in contact with the pomalidomide orlenalidomide, to produce a first expanded CD34⁺ hematopoietic stem cellpopulation; wherein said factors comprise SCF, Flt-3 and IL-3, (b)expanding the first expanded CD34⁺ hematopoietic stem cell population ina medium in contact with one or more factors in the presence of aplurality of feeder cells, wherein said feeder cells are adherentplacental stem cells, to produce a second expanded human CD34⁺hematopoietic stem cell population; wherein said second population ofhuman CD34⁺ hematopoietic stem cells differentiate into erythrocytesduring said expanding, wherein said factors comprise SCF, Epo andInsulin-like growth factor I (IGF-1), and (d) isolating saiderythrocytes from said second expanded hematopoietic stem cellpopulation.
 2. The method of claim 1, wherein said hematopoietic stemcells are obtained from a source selected from the group consisting ofumbilical cord blood, placental blood, peripheral blood, and bonemarrow.
 3. The method of claim 1, wherein said hematopoietic stem cellsare obtained from placental perfusate.
 4. The method of claim 1, whereinsaid hematopoietic stem cells are obtained from umbilical cord blood andplacental perfusate.
 5. The method of claim 4, wherein said placentalperfusate is obtained by passage of perfusion solution through only thevasculature of a placenta.
 6. The method of claim 3, wherein saidplacental perfusate is obtained by passage of perfusion solution throughonly the vasculature of a placenta.
 7. The method of claim 1, whereinsaid feeder cells are from the same individual as said hematopoieticstem cells.
 8. The method of claim 1, wherein said feeder cells are froma different individual than said hematopoietic stem cells.
 9. The methodof claim 1, wherein said adherent placental stem cells are selected fromthe group consisting of: (a) CD200⁺ or HLA-G⁺; (b) CD73⁺, CD105⁺, andCD200⁺; (c) CD200⁺ and OCT-4⁺; (d) CD73⁺, CD105⁺ and HLA-G⁺; (e) CD10⁺,CD34⁻, CD105⁺, and CD200⁺; (f) HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻; (g)CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻; (h) CD10⁻, CD33⁻, CD44⁺,CD45⁻, and CD117⁻; (i) HLA A,B,C⁺, CD45⁻, CD34⁻, CD133⁻; (j) positivefor CD10, CD13, CD38, CD44, CD90, CD105, and are one or more of CD200positive, HLA-G positive, and CD117 negative; (k) CD200⁺ and CD10⁺, asdetermined by antibody binding, and CD117⁻, as determined by bothantibody binding and RT-PCR; and (l) CD10⁺, CD29⁻, CD54⁺, CD200⁺,HLA-G⁺, HLA class I⁺ and β-2-microglobulin⁺.
 10. The method of claim 1,wherein a plurality of said hematopoietic stem cells has a blood typeselected from the group consisting of blood type A, blood type B, bloodtype AB, and blood type O, wherein a plurality of said hematopoieticstem cells is Rh positive or Rh negative, and wherein a plurality ofsaid hematopoietic stem cells optionally has a blood type selected fromthe group consisting of-blood type M, blood type N, blood type S, bloodtype s, blood type P1, blood type Lua, blood type Lub, blood type Lu(a),blood type K (Kell), blood type k (cellano), blood type Kpa, blood typeKpb, blood type K(a+), blood type Kp(a−b−), blood type K− k− Kp(a−b−),blood type Le(a−b−), blood type Le(a+b−), blood type Le(a−b+), bloodtype Fy a, blood type Fy b, blood type Fy(a−b−), blood type Jk(a−b−),blood type Jk(a+b−), blood type Jk(a−b+), and blood type Jk(a+b+). 11.The method of claim 10, wherein the hematopoietic stem cells are type O,Rh positive; type O, Rh negative; type A, Rh positive; type A, Rhnegative; type B, Rh positive; type B, Rh negative; type AB, Rh positiveor type AB, Rh negative.